The present invention describes a methodology for the use of glycosaminoglycan degrading enzymes to modulate events in the wound healing process.
Growth factors are naturally occurring polypeptides that elicit hormone type modulation of cell proliferation and differentiation. The mechanism by which these events transpire is typically initiated by the growth factor contacting specific receptors or receptor systems which are located on the cell surface. The sequence of intracellular events that occur subsequent to the receptor/growth factor interaction are responsible for mitogenic and differentiating responses by the cell. These mechanisms are not fully understood but may include activation of tyrosine kinases, nucleotide metabolism and variations in cell electrolyte levels (Burgess and Macaig, Ann. Rev. Biochem, 58:575-606, 1989).
For most cell types, events of mitogenesis and differentiation are subdued in the normal adult animal. These growth factor mediated events are more commonly associated with developing organisms, during wound healing processes or in various disease states including cancer and vascular disease. For example, the normal turnover rate of endothelial cells, including the lining of microvessels and arteries, is measured in thousands of days. During normal wound healing however, these endothelial cells proliferate rapidly, with a turnover rate of approximately five days (Folkman and Shing, J. Biol. Chem. 267(16):10931-10934, 1992). The increase in proliferation that occurs during wound healing appears to be the result of an increase in the local concentration of various angiogenic molecules, including growth factors.
The fibroblast growth factor family includes at least seven polypeptides that have been shown to stimulate proliferation in various cell lines including endothelial cells, fibroblasts, smooth muscle cells and epidermal cells. Included in this group are acidic fibroblast growth factor (FGF-1), basic fibroblast growth factor (FGF-2), int-2 (FGF-3), Kaposi sarcoma growth factor (FGF-4), hst-1 (FGF-5), hst-2 (FGF-6) and keratinocyte growth factor; (FGF-7) (Baird and Klagsbrun, Ann. N.Y. Acad. Sci. 638: xiv, 1991). These molecules, and other cytokines including tissue growth factors, TGF.alpha. and TGF.beta., platelet derived growth factors, PDGF, granulocyte-macrophage colony stimulating factor, GM-CSF, interleukin 3, IL-3, and platelet factor 4, PF4, share a common feature in their affinity for heparin (Clark, Dermatol. Clin. 11:647-666, 1993). Specific cell type responses also have been associated with particular factors. EGF and TGF.alpha. stimulate the proliferation of keratinocytes, TGF.beta. stimulates collagen and fibronectin synthesis, PDGF stimulates angiogenesis and granulation tissue formation and FGF-7 stimulates epithelial cell proliferation (Staiano-Coico, et al., J. Exp. Med. 178:865-878, 1993). PDGF, FGF-2 and a recently described heparin binding epidermal growth factor HB-EGF (Higashiyama, et. al., Science 251:936-939, 1991) additionally are involved in the proliferation and migration of vascular smooth muscle cells and vascular endothelial cells.
The change in a cell's metabolic state from quiescent to proliferative or migratory implies an enhanced availability of the appropriate signaling molecules in the vicinity of the cell. In principle this could result from either an increase in growth factor synthesis or the release of growth factors from storage reservoirs. In nature, both mechanisms have been observed. The expression of FGF-1, FGF-2, FGF-5 and FGF-7 are upregulated after full thickness dermal injury (Werner, et. al., Proc. Natl. Acad. Sci. 89:6896) while TGF.beta., FGF-2 and PDGF synthesis increases in smooth muscle cells in response to vascular injury. Growth factors also have been detected in most solid tissues extracted from normal adult, non-wounded samples. Despite the presence of growth factors in these areas, the cells comprising them are not in a proliferative state. Apparently, growth factors are stored outside the cell in basement membranes and the extracellular matrix where they are prevented from contacting their respective cell surface receptors. In this mode they serve as an emergency supply for wound repair and blood vessel formation functions (Vlodavsky, et. al. TIBS 16:268-271, 1991).
An initial event in tissue or vessel injury may involve a mechanical dislodging of growth factors from the extracellular space, making them available to cell surface receptors where they stimulate cell proliferation and cell synthesis of additional growth factors. Alternately, cells under stress may secrete molecules which displace the extracellular growth factors from these storage reservoirs. Tumor cells have been shown to secrete degradative enzymes, including proteoglycanases, collagenases and metalloproteinases, coincident with metastasis (Nicolson Curr. Opinion Cell Biol. 1:1009-1019, 1989). In addition to facilitating tumor migration through blood vessels, the destruction of extracellular matrix components releases growth factors, thereby promoting new blood vessel formation which feeds the growing tumor mass (Folkman, et al., Am J Pathol 130:393-400, 1988).
Extracellular matrices (ECM) are multi-component structures synthesized by and surrounding various cell types including endothelial, epithelial, epidermal and muscle cells. The ECM is formed largely of collagen and heparan sulfate proteoglycans. It also contains fibronectin, chondroitin sulfate proteoglycans and smaller proteins. Growth factors are sequestered in these matrices by association with the glycosaminoglycan portion of the heparan sulfate proteoglycans. Heparin and heparan sulfate are polysaccharides formed of alternating hexuronic, either D-glucuronic or L-iduronic, and glucosamine, N-acetylated or N-sulfated, residues with varying sulfation patterns. Heparin extracted from porcine intestines, bovine lungs or human mast cells displays a high degree of sulfation, up to 2.6 sulfates per disaccharide unit, and a greater iduronic acid content than heparan sulfate. Conversely, heparan sulfate has a lower degree of sulfation and preferentially contains glucuronic acid in the alternating saccharide position. "Heparin like" regions of high iduronic acid and high sulfation have been associated with the bFGF binding region of heparan sulfate from human fibroblasts (Turnbull, et al., J. Biol. Chem. 267(15) 10337-10341, 1992). However, the composition of heparan sulfate in the extracellular matrix has not been fully characterized.
The stimulation of cell proliferation and migration by growth factors constitutes one of the events in the wound healing process which is a multifactoral interactive process involving biochemical mediators, the extracellular matrix and parenchymal cells. The wound healing process is generally divided into three temporally overlapping phases: inflammation, proliferation and remodeling. During inflammation, blood borne cells infiltrate the wound site and release several mediating molecules including platelet derived growth factor, von Willibrand factor, thrombospondin, fibronectin, fibrinogen, 5-hydroxytryptophan, thromboxane-A2 and adenosine diphosphate (Kirsner and Eaglstein, J. Dermatol. 151:629-640, 1993). A platelet plug and thrombus are formed and provide a matrix for monocytes, fibroblasts and keratinocytes. Chemotactic molecules attract monocytes which transform into macrophages and secrete additional growth factors (Nathan and Sporn, J. Cell Biol. 113:981-986, 1991). Neutrophils may assist in this process by secreting the degradative enzymes elastase and collagenase which enhance the passage of cells through the basement membranes.
Keratinocytes and epidermal cells, which are involved in the closure of dermal wounds, migrate to the wound site during the proliferative phase. Angiogenesis, the formation of new blood vessels in response to chemoattractant and angiogenic signals (Folkman and Klagsbrun, Science 235:442-447, 1987), and fibroplasia, the accumulation of fibroblasts and formation of granulation tissue, also occurs during the proliferative phase. Tissue remodeling is accompanied by the secretion of matrix components, including fibronectin, collagen and proteoglycans which serve as a scaffold for cellular migration and tissue support. Type III collagen, synthesized in the earlier stages of wound healing, is replaced by the more permanent type I form through a process of proteolytic turnover.
Ischemia refers to the pathological condition due to the localized dysfunction of the vascular system resulting in inadequate blood supply with subsequent tissue damage. In this case revascularization, whether through the stimulation of angiogenesis or by surgical methods, must precede the normal wound healing course of the damaged tissue.
The action of enzymes which degrade components of the extracellular matrix and basement membranes may facilitate the events of tissue repair by a variety of mechanisms including the release of bound cytokines entrapped by heparan sulfate and by increasing the permeability of the matrix, thereby enhancing the mobility of mediator molecules, growth factors and chemotactic agents, as well as the cells involved in the healing process. Glycosaminoglycans are subject to degradation by a variety of eukaryotic and prokaryotic enzymes. Heparan sulfate degrading activity has been detected in platelets (oldberg et al. Biochemistry, 19:5755-5762, 1980), tumor cells (Nakajima, et al. J. Biol. Chem. 259:2283-2290, 1984) and endothelial cells (Gaal et al. Biochem. Biophys. Res. Comm., 161:604-614, 1989). These heparanase enzymes act by catalyzing the hydrolysis of the carbohydrate backbone of heparan sulfate at the hexuronic acid (1.fwdarw.4) glucosamine linkage (Nakajima et al., J. Cell, Biochem., 36:157-167, 1988). Mammalian heparanases are typically inhibited by the highly sulfated heparin form of the heparin-heparan sulfate family. However, accurate biochemical characterizations of these enzymes have thus far been prevented by the lack of a method to obtain homogeneous preparations of the molecules.
Heparin degrading enzymes also have been found in microorganisms including Flavobacterium heparinum (Lohse and Linhardt, J. Biol. Chem. 267:2437-24355, 1992), Bacteroides strains (Saylers, et al., Appl. Environ. Microbiol. 33:319-322, 1977; Nakamura, et al., J. Clin. Microbiol. 26:1070-1071, 1988), Flavobacterium Hp206 (Yoshida, et al., 10th Annual Symposium of Glycoconjugates, Jerusalem 1989) and Cytophagia species (Bohn, et al., Drug Res. 41(I), Nr. 4:456-460, 1991). Chrondoitin sulfate degrading enzymes have been isolated from several microorganisms including Flavobacterium heparinum (Michaleacci, et al., Biochem. J. 151:123, 1975), Bacteroides species (Saylers, et al. J. Bacteriol. 143:781, 1980; Linn, et al., J. Bacteriol. 156:859, 1983; Steffen, et al., J. Clin. Microbiol. 14:153, 1981), Proteus vulgaris (Uamagata, et al., J. Biol. Chem. 243:1523, 1968, Suzuki, Meth. Enzymol. 28:911, 1972), Beneckea, Microcossus and Vibrio species (Kitamikada and Lee, Appl. Microbiol. 29:414, 1975) and Arthrobacter aurescens (Hiyam and Okada, J. Biol. Chem. 250:1824-1828, 1975).
F. heparinum produces three forms of heparinase, heparinase 1, heparinase 2, and heparinase 3 (heparitinase) (Lohse and Linhardt, J. Biol. Chem. 267:24347-24355, 1992). All three enzymes cleave at glucosamine (1.fwdarw.4) hexuronic acid linkages with differing degrees of specificity depending on sulfation patterns and particular hexuronic acid residue, iduronic or glucuronic, in a particular cleavage site (Desai, et al., Arch. Biochem. Biophys. 306:461-468, 1993). F. heparinum also produces two enzymes which degrade members of the chondroitin sulfate/dermatan sulfate family. These are chondroitin lyase AC, which degrades both chondroitin sulfate A and chondroitin sulfate C by cleaving the galactosamine (1.fwdarw.4) glucuronic acid linkage in the polysaccharide backbone and chondroitin lyase B which degrades dermatan sulfate (chondroitin sulfate B) by cleaving the galactosamine (1.fwdarw.4) iduronic acid linkage in the polysaccharide backbone. The enzymatic mechanism of the F. heparinum enzymes is through an elimination reaction, thereby differentiating them from the mammalian glycosaminoglycan degrading enzymes. Furthermore, none of the F. heparinum lyase enzymes appear inhibited by glycosaminoglycan molecules as are the mammalian enzymes.
Mammalian heparanase, partially purified from tumor cell line extracts, as well as heparinase 1 and heparinase 3 from Flavobacterium heparinum, have been shown to release .sup.125 I radiolabelled FGF-2 that had been pre-adsorbed to extracellular matrix synthesized in vitro by bovine aorta endothelial cells (Bashkin, et al. J. Cell. Physiol. 167:126-137, 1992). However, since unfractionated and low molecular weight heparin elicited a similar release of the exogenously absorbed .sup.125 I radiolabelled FGF-2, it is not clear from these reports whether the measured release was due to the enzymatic degradation of the heparan sulfate in the ECM or an ion exchange type electrolytic displacement of FGF-2 from the negatively charged heparan sulfate. The same research group reported the release of growth promoting activity from vascular smooth muscle cells by treatment with heparinase 3 and from extracellular matrix by exposure to extracts of neutrophils or lymphoma cells. However, there has been no demonstration of the release of growth promoting activity from extracellular matrix by contact with bacterial glycosaminoglycan degrading enzymes nor have these enzymes been shown to promote tissue repair or new vessel growth in vivo.
It is therefore an object of the present invention to provide a method and compositions for enhancing and controlling tissue repair and new vessel growth.
It is a further object of the present invention to provide highly purified glycosaminoglycan degrading enzyme pharmaceutical compositions for use in enhancement of tissue repair and manipulation of angiogenesis.